Evaluation of the wound age and vitality is a persistent problem for forensic examinations to determine when injuries occurred and whether during the antemortem or post-mortem period. This study aimed to use the inflammatory cells and mediators as a target marker to determine wound age in antemortem or post-mortem samples in rats. Wound skin samples (n=39) were collected from antemortem groups aged 30, 60, 180, and 360 min and post-mortem groups within the same periods with control samples (unwounded group). Immunohistochemical staining examined all tissue samples for two inflammatory cell markers (CD15, CD68) and 2 inflammatory mediators (IL-6, TNF-α). The results showed that CD15-positive neutrophils were expressed in all time intervals of antemortem, and the expression gradually increased from 30 to 360 min. The CD68-positive macrophages began at 30 minutes and reached the highest level at 360 minutes in the antemortem wound. Because of the negative expression of CD15 and CD68 in all postmortem groups, they could be a marker for comparing the vital and non-vital wounds. Furthermore, the Immunoreactivity results showed that the IL-6 and TNF-α were negatively expressed in unwounded skin samples. In addition, the periods after the induced wound produced a rise in the expression area of both markers in antemortem wounds. In this study, IL-6 and TNF-α expressions were negative or low in areas far from wound margin, unwounded specimens, and post-mortem wounds. CD15, CD68, IL-6, and TNF-α may be valuable markers for assessing wound age and vitality.